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In sporadic breast cancer, no mutations of the BRCA1 gene have been reported so far, whereas BRCA1 mRNA is markedly decreased in invasive breast cancer. To elucidate the contribution of the BRCA1 gene in sporadic breast cancer, we quantified the BRCA1 protein, using [ 125 I] labeling of wholecell proteins, lentil-lectin affinity chromatography, immunoprecitation by anti-BRCA1 antibodies (C-20, D-20, I-20 and K-18), purification of the immune complex by protein A affinity chromatography and chromato-focusing. As loss of 1 allele may lead to a decreased expression of the gene, 10 tumors were previously checked for loss of heterozygosity (LOH) of the BRCA1 gene, using 3 intragenic microsatellite markers. Our results indicated that the BRCA1 gene product was decreased in the 4 tumors with LOH compared with matched normal breast tissues. Reduced amounts of BRCA1 protein were also detected in 3 of 6 tumors without LOH. Our quantitative method allowed us to demonstrate that the BRCA1 protein level was decreased in sporadic invasive breast carcinomas with or without LOH of the BRCA1 gene, implying multiple mechanisms of BRCA1 expression downregulation in these tumors. Our data suggest that the amount of BRCA1 protein present may play an important role in human sporadic breast carcinoma. Int. J. Cancer 80:823-826, 1999. Wiley-Liss, Inc.While familial breast cancer or familial breast/ovarian cancer often accompanies germ-line mutations of the BRCA1 gene (Miki et al., 1994), there is no evidence of BRCA1 mutations in sporadic breast cancer and BRCA1 mutations in sporadic ovarian cancer are rare (Futreal et al., 1994, Merajver et al., 1995. However BRCA1 gene transcription is markedly reduced in invasive breast tumors relative to normal breast epithelium, ductal carcinoma in situ (Thompson et al., 1995) and breast cancer cell lines (Ribieras et al., 1997), suggesting a role for BRCA1 in sporadic tumors. So far, BRCA1 gene expression in tumor tissues and cell lines has been monitored mainly at the mRNA level, by either Northern blot (Marquis et al., 1995), ribonuclease protection assay (Thompson et al., 1995) or competitive RT-PCR (Ribieras et al., 1997). In this study, we evaluated the relative amount of BRCA1 protein in ductal invasive carcinomas, using a strategy of biochemical purification previously devised in our laboratory for measureing HLA-DR antigen amounts (Bernard et al., 1984(Bernard et al., , 1985 and adapted for the BRCA1 protein (Bernard-Gallon et al., 1997).As loss of 1 allele might lead to reduced expression of the corresponding gene, we searched for loss of heterozygosity (LOH) of the BRCA1 gene, using 3 intragenic microsatellite markers. The BRCA1 protein was then quantified in the 2 groups of tumors, with and without LOH, and compared in tumors and normal matched tissues. Our results show that the amount of BRCA1 protein was decreased in all tumors with LOH as well as in a majority of ductal invasive carcinomas without LOH. MATERIAL AND METHODS Patient samplesTen sporadic invasive ductal breast carcinomas...

To enhance affinity for malignant cartilaginous tumors (chondrosarcomas), quaternary ammonium (QA) conjugates of chlorambucil and melphalan were prepared by linking the QA moiety to nitrogen mustards via an amide bond. They exhibited closely similar and sometimes more favorable values than their parent compounds. In the cell lines tested, the two QA conjugates displayed appreciable cytotoxicity, the QA conjugate of chlorambucil even showing an enhanced efficiency against chondrosarcoma compared with chlorambucil.

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As part of a cartilage targeting program based on the affinity of the quaternary ammonium (QA) moiety for cartilage, QA derivatives of D-glucosamine (DG), an antirheumatic drug exhibiting a natural tropism for cartilaginous tissues, were designed and evaluated by pharmacokinetic studies. Two QA-DG conjugates were synthesized and labeled with (14)C by cross-linking the QA entity (trimethylammonium or pyridinium) to [(14)C]DG via an amide bond in a two-step procedure. After intravenous injection to male Sprague-Dawley rats, the two (14)C-labeled conjugates exhibited similar pharmacokinetic profiles, but their behavior clearly differed from that of unconjugated DG in several ways. (i) The tissue distribution for the conjugates was more restricted, with a decreased radioactivity level for whole tissues except for kidney, cartilage, and skin. (ii) The radioactivity concentrated more rapidly and strongly in cartilage for the conjugates than for DG for the short times after injection; on the other hand, 1 h after administration, the radioactivity level in cartilage was higher for DG, this result being consistent with the tropism already observed for this compound. (iii) Both conjugates were eliminated predominantly by the urinary route (85%); the radioactivity level in urine for DG was lower (45% of the injected dose), and significant (14)CO(2) was found in expired air, indicating metabolization and utilization of DG for energy-consuming processes. (iv) Blood and plasma kinetics studies displayed an enterohepatic cycle for DG, whereas for the QA conjugates, a rapid disappearance was observed. (v) HPLC analyses of plasma and urine indicated a low degree of metabolization for the conjugates, most of the radioactivity recovered in urine and plasma corresponding to the unchanged molecule. This study demonstrates that the introduction of the QA moiety on DG modifies its biodistribution and lends it a greater specificity for cartilage, at least for short times after injection. These findings justify further work on QA derivatives of other antirheumatic agents.

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Current evidence strongly supports a role for the breast tumour suppressor genes, BRCA1 and BRCA2, in both normal development and carcinogenesis. In vitro observations reported that BRCA1 and BRCA2 are expressed in a cell cycle-dependent manner. Interestingly, differences in the actions of n-3 and n-6 polyunsaturated fatty acids have been observed: while the n-3 polyunsaturated fatty acids have been described to reduce pathological cell growth, the n-6 polyunsaturated fatty acids have been found to induce tumour proliferation. Here, we examined the expression of BRCA1 and BRCA2 in breast cell lines after treatment with polyunsaturated fatty acids. Real-time quantitative polymerase chain reaction determinations conclusively demonstrated increases in BRCA1 and BRCA2 mRNA expressions in MCF7 and MDA-MB 231 tumour cell lines after treatment with n-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid), but no variation was noticed with the n-6 polyunsaturated fatty acid (arachidonic acid). On the other hand, no variation of the expression of BRCA1 and BRCA2 mRNA was detected in MCF10a normal breast cell line treated by polyunsaturated fatty acids. The level of BRCA1 and BRCA2 proteins quantified by affinity chromatography remained unchanged in tumour (MCF7, MDA-MB 231) and normal (MCF10a) breast cell lines. We suggest the presence of a possible transcriptional or post-transcriptional regulation of BRCA1 and BRCA2 after n-3 polyunsaturated fatty acid treatment in breast tumour cells.